PPT 206 Instrumentation, Measurement and Control SEM 2 (2012/2013) Dr. Hayder Kh. Q. Ali 1

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5 Essentially, the fluidics system consists of a central channel/core through which the sample is injected, enclosed by an outer sheath that contains faster flowing fluid. As the sheath fluid moves, it creates a massive drag effect on the narrowing central chamber. This alters the velocity of the central fluid whose flow front becomes parabolic with greatest velocity at its center and zero velocity at the wall (see Figure 1). Under optimal conditions (laminar flow) the fluid in the central chamber will not mix with the sheath fluid. The flow characteristics of the central fluid can be estimated using Reynolds Number (Re). Without hydrodynamic focusing the nozzle of the instrument would become blocked, and it would not be possible to analyze one cell at a time

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8 Light that is scattered in the forward direction, typically up to 20º offset from the laser beam’s axis, is collected by a lens known as the forward scatter channel (FSC). The FSC intensity roughly equates to the particle’s size and can also be used to distinguish between cellular debris and living cells. Light measured approximately at a 90° angle to the excitation line is called side scatter. The side scatter channel (SSC) provides information about the granular content within a particle. Both FSC and SSC are unique for every particle, and a combination of the two may be used to differentiate different cell types in a heterogeneous sample.

9 Fluorescence measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome- labeled cell surface receptors or intracellular molecules such as DNA and cytokines. Flow cytometers use separate fluorescence (FL-) channels to detect light emitted. The number of detectors will vary according to the machine and its manufacturer. Detectors are either silicon photodiodes or photomultiplier tubes (PMTs). Silicon photodiodes are usually used to measure forward scatter when the signal is strong. PMTs are more sensitive instruments and are ideal for scatter and fluorescence readings.

10 The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting (passing) others. There are three major filter types. ‘Long pass’ filters allow through light above a cut-off wavelength, ‘short pass’ permit light below a cut-off wavelength and ‘band pass’ transmit light within a specified narrow range of wavelengths (termed a band width). All these filters block light by absorption (Figure 2).

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12 When a filter is placed at a 45º angle to the oncoming light it becomes a dichroic filter/mirror. As the name suggests, this type of filter performs two functions, first, to pass specified wavelengths in the forward direction and, second, to deflect blocked light at a 90° angle. To detect multiple signals simultaneously, the precise choice and order of optical filters will be an important consideration (refer to Figure 3).

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17 The speed of flow sorting depends on several factors including particle size and the rate of droplet formation. A typical nozzle is between 50–70 µM in diameter and, depending on the jet velocity from it, can produce 30,000–100,000 droplets per second, which is ideal for accurate sorting. Higher jet velocities risk the nozzle becoming blocked and will also decrease the purity of the preparation.

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19 Light is a form of electromagnetic energy that travels in waves. These waves have both frequency and length, the latter of which determines the color of light. The light that can be visualized by the human eye represents a narrow wavelength band (380–700 nm) between ultraviolet (UV) and infrared (IR) radiation (Figure 5). The visible spectrum can further be subdivided according to color, standing for red, orange, yellow, green, blue and violet. Red light is at the longer wavelength end (lower energy) and violet light at the shorter wavelength end (higher energy).

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23 As E emission contains less energy than was originally put into the fluorochrome it appears as a different color of light to E excitation. Therefore, the emission wavelength of any fluorochrome will always be longer than its excitation wavelength. The difference between E excitation and E emission is called Stokes Shift and this wavelength value essentially determines how good a fluorochrome is for fluorescence studies. After all, it is imperative that the light produced by emission can be distinguished from the light used for excitation. This difference is easier to detect when fluorescent molecules have a large Stokes Shift.

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25 Maximal absorbance usually defines the laser spectral line that is used for excitation. In the case of FITC, its maximum falls within the blue spectrum. Therefore, the blue Argon-ion laser is commonly used for this fluorochrome, as it excites at 488 nm, close to FITC’s absorbance peak of 490 nm. FITC emits fluorescence over the range 475–700 nm peaking at 525 nm, which falls in the green spectrum. If filters are used to screen out all light other than that measured at the maximum via channel A (see Figure 7), FITC will appear green. Hence, ‘fluorescence color’ usually refers to the color of light a fluorochrome emits at its highest stable excited state. However, if FITC fluorescence is detected only via channel B (see Figure 7), it will appear orange and be much weaker in intensity. How the flow cytometer is set up to measure fluorescence will ultimately determine the color of a fluorochrome.

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29 Instead, a process called fluorescence compensation is applied during data analysis, which calculates how much interference (as a %) a fluorochrome will have in a channel that was not assigned specifically to measure it. Figure 8 helps to explain the concept. The graphs show the emission profiles of two imaginary fluorochromes ‘A’ and ‘B’ which are being detected in FL-1 and FL-2 channels respectively. Because the emission profiles are so close together, a portion of fluorochrome A spills over into FL-2 (red shade) and conversely, some of fluorochrome B reaches FL-1 (dark blue shade).

30 To calculate how much compensation needs to be applied to the dataset if both dyes are used simultaneously, some control readings must first be taken. Fluorochrome A should be run through the flow cytometer on its own and the % of its total emission that is detectable in FL-2 (spillover) determined. The procedure should be repeated with fluorochrome B, except that this time FL-1 is spillover. Suppose the results are:

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35 Contour diagrams are an alternative way to demonstrate the same data. Joined lines represent similar numbers of cells. The graph takes on the appearance of a geographical survey map, which, in principle, closely resembles the density plot. It is a matter of preference but sometimes discreet populations of cells are easier to visualize on contour diagrams e.g. compare monocytes in Figure 9.

36 On the density plot, each dot or point represents an individual cell that has passed through the instrument. Yellow/green hotspots indicate large numbers of events resulting from discreet populations of cells. The colors give the graph a three-dimensional feel. After a little experience, discerning the various subtypes of blood cells is relatively straightforward.

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39 Ideally, flow cytometry will produce a single distinct peak that can be interpreted as the positive dataset. However, in many situations, flow analysis is performed on a mixed population of cells resulting in several peaks on the histogram. In order to identify the positive dataset, flow cytometry should be repeated in the presence of an appropriate negative isotype control (see Figure 12).

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44 The differences between the blood profiles of a healthy individual and one suffering from leukemia, for instance, are very dramatic. This can be seen from the FSC v SSC plots in Figure 16. In the healthy person the cell types are clearly defined, whereas blood from a leukemia patient is abnormal and does not follow the classic profile.

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